Artificial biological membranes have been used for a variety of applications including for medical and industrial applications. Artificial membranes are known to those skilled in the art, and include a mixture of collagen and elastin (U.S. Pat. No. 5,416,074), an amphiphilic compound (e.g., a phospholipid or other purified membrane constituent; U.S. Pat. No. 4,931,498), or supported phospholipid/alkanethiol bilayers ("supported hybrid bilayer membranes) comprising a phospholipid monolayer formed onto a alkanethiol monolayer. Regarding the latter, it has been described previously that when alkanethiols are applied to a thin film of gold, a self-assembled monolayer of alkanethiol is formed by adsorption of the alkanethiols to the surface of the gold (see, e.g., Nuzzo et al., 1987, J. Am. Chem. Soc., 109:2358-68; Prime and Whiteside, 1991, Science, 252:1164-67). Such self-assembled monolayers have been used as a surface onto which fibronectin is adsorbed in forming a tissue culture substrate (Mrksich et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10775-8); and as a tetracyanoquinodimethane mediated biosensor for glucose (Pandey et al., 1995, Biosens. Bioelectron., 10:669-74).
Biological (cell) membranes, as they exist in nature, differ from artificial membranes in several respects. For example, cell membranes have active transport systems for macromolecules including cations, proteins, and DNA; whereas lipid bilayers alone lack such transport systems. Additionally, typical artificial membranes comprise phospholipid-molecules only; whereas biological membranes include an ordered architecture of phospholipids, and membrane-associated proteins such as enzymes and receptor molecules. While a lipid bilayer can be formed by the addition of phospholipid vesicles to alkanethiol monolayer on gold, at the time of the invention it was not believed that this method could be applied to cell membranes. This is because lipid vesicle spreading and advancement over the alkanethiol monolayer requires the surface of the monolayer to be attractive to the vesicles, thereby promoting the hydrophobic interaction between the alkyl chains of the alkanethiols and the phospholipid molecules. However, the architecture and multi-component composition of biological membranes present steric hindrances to the interaction necessary for phospholipid vesicles to spread and advance over, and immobilize to, the alkanethiol monolayers. Such steric hindrances are repulsive and destabilizing forces thought to help oppose the attractive interaction between phospholipids and alkanethiol monolayer, and thereby would preclude the formation of a stable association, and resultant immobilization, between phospholipids and an alkanethiol monolayer on gold. Moreover, biological membranes typically comprise a multitude of proteins with extramembrane domains (e.g., protein segments protruding from the portion of the membrane that faces the cell cytoplasm, and protein segments exposed to the portion of the cell membrane that faces extracellularly). However, a multitude of proteins has not been observed to be adsorbed onto alkanethiol monolayers (Prime and Whitesides, 1991, supra). It has also been demonstrated that the phospholipid/alkanethiol bilayer shows little nonspecific adsorption of protein, and thus prevents the adsorption of proteins onto either the alkanethiol monolayer or the metal film of the support (Plant et al., 1995, Anal. Biochem. 226:342-348). Further, the close association between the thiol sulfur and gold of the alkanethiol monolayer may preclude the normal insertion of a transmembrane protein (Plant et al., 1994, Biophys. J. 67:1126-33).
Additionally, membrane-associated proteins represent sensing and recognition molecules residing in biological membranes. It is appreciated by those skilled in the art that such membrane-associated proteins lose their specificity or activity (e.g. enzymatic activity) upon slight alterations of their lipid environment (e.g., membrane depolarization) and/or upon alterations in the membrane's ordered architecture (see, e.g., Brenner et al., 1977, Can. J. Biochem. 55:126-33; Reifarth et al., 1997, J. Membr. Biol. 155:95-104). For example, free fatty acids, and related compounds such as aliphatic aldehydes, have been shown to inhibit cell membrane-associated enzymatic activity by perturbing the lipid bilayer and disturbing protein-membrane lipid interactions of cell membranes (Lapshina et al., 1995, Scand. J. Clin. Lab. Invest. 55:391-7). Therefore, any attempt to immobilize a biological membrane could potentially affect the activity inherent to components of the membrane in its natural setting.
Thus, because natural biological (cell) membranes differ from artificial membranes, and to date only artificial membranes have been immobilized, there remains a need for methods to immobilize biological membranes. Such a method would obviate the current and tedious approach of isolating membrane proteins, and attempting to reconstitute lipid bilayers with such isolated proteins.